ACCELERATED PAPER: Identification of human tumour suppressor genes by monochromosome transfer: rapid growth-arrest response mapped to 9p21 is mediated solely by the cyclin-D-dependent kinase inhibitor gene, CDKN2A(p16INK4A)

Microcell transfer of intact normal human chromosomes into immortalmouse and hamster fibroblast cell lines has revealed growthsuppressive activity associated with a small sub-set of thehuman complement. Here, we describe the results of a detailedstudy aimed at identifying the gene or genes responsible forthe rapid growth-arrest response obtained with human chromosome-9.Initially, STS-PCR deletion mapping of segregants arising inmonochromosome transfer experiments was used successfully tolocalize the active sub-chromosomal region to 9p21. Subsequentfine structure deletion mapping of previously uninformativehybrid segregants, employing additional markers between D9S162and D9S171, provided strong evidence that the cyclin-dependentkinase (cdk) inhibitor gene CDKN2A (p16^INK4A) was solely responsiblefor the chromosome-9 effect; 9p21 microdeletions in a significantproportion of segregant clones were restricted to a single CDKN2Aexon. Transfection experiments with CDKN2A and CDKN2B cDNA expressionvectors, using mouse A9 cells and three human malignant melanomacell lines as recipients, provided further evidence in supportof this hypothesis. Collectively, our results indicated thatexpression of human CDKN2A (controlled either by its naturalregulatory elements, or by a cytomegalovirus promoter) is incompatiblewith in vitro proliferation in immortalized rodent cells andin human melanoma cell lines. The rapidity of the growth inhibitoryeffects of CDKN2A was inconsistent with a mode of action involvinginduction of replicative cell senescence via telomerase repression,but was consistent with a mechanism based on cell cycle arrestthrough cdk inhibition. The study described here has generateda panel of microdeleted monochromosome-9 donor hybrids whichmay prove valuable in functional investigations aimed at identifyingother important tumour suppressor genes located on human chromosome-9.     

吲哚-3-乙酸对人外周血淋巴细胞微核和SCE频率的影响

背景与目的:研究吲哚-3-乙酸对人体外周血淋巴细胞微核和SCE频率的影响.材料与方法:应用人体外周血淋巴细胞测定吲哚-3-乙酸诱导微核形成率试验和姊妹染色单体互换率(SCE).结果:各吲哚-3-乙酸处理组与阴性对照组相比,微核形成率和SCE差异均存在显著性.结论:吲哚-3-乙酸对人外周血淋巴细胞的遗传物质具有损伤作用.     

Development of a novel enzyme/prodrug combination for gene therapy of cancer: horseradish peroxidase/indole-3-acetic acid

This paper demonstrates the potential for utilizing the plant enzyme, horseradish peroxidase (HRP), in a gene-directed enzyme prodrug therapy context. Human T24 bladder carcinoma cells transfected with a mammalian expression vector containing the HRP cDNA were selectively sensitized to the nontoxic plant hormone, indole-3-acetic acid (IAA). The HRP/IAA-induced cell kill was effective in normoxic and anoxic conditions. The activated drug is a long-lived species able to cross cell membranes, and cell contact appears not to be required for a bystander effect to take place. These preliminary results suggest that the delivery of the HRP gene to human tumors followed by IAA treatment may provide a novel cancer gene-directed enzyme prodrug therapy approach, with potential to target hypoxic cells.     

Analysis of the horseradish peroxidase/indole-3-acetic acid combination in a three-dimensional tumor model

Horseradish peroxidase has previously been shown to catalyze the conversion of indole-3-acetic acid (IAA) to a potent cytotoxin in a gene therapy setting. A three-dimensional spheroid model composed of a human head and neck carcinoma cell line, has been used to mimic the tumor microenvironment, such as regions of hypoxia. Exposure of intact spheroids to 0.05-5 mM concentrations of IAA and the halogenated indole, 5-bromoindole-3-acetic acid (5Br-IAA), resulted in decreased cell survival, and demonstrates that this combination is effective under tumor-simulated conditions. In addition, 5Br-IAA, displayed selectivity for spheroids with a large hypoxic fraction following short exposure times.     

Ellagic acid: a potent naturally occurring inhibitor of benzo[a]pyrene metabolism and its subsequent glucuronidation, sulfation and covalent binding to DNA in cultured BALB/C mouse keratinocytes

The metabolism of [³H]benzo[a]pyrene (BP) by cultured primarykeratinocytes prepared from BALB/C mouse epidermis was foundto be largely inhibited by the dietary plant phenol, ellagicacid. Varying concentrations of ellagic acid added to the keratinocytecultures resulted in a dosedependent inhibition of the cytochromeP-450-dependent monooxygenases aryl hydrocarbon hydroxylase(AHH) and 7-ethoxycoumarin-0-deethylase (ECD). The major organicsolvent-extractable metabolites found intracellularly in thecultured cells were trans-7,8-dihydro-7,8-dihydroxybenzo[a]-pyrene(BP-7,8-diol) and 3-hydroxybenzo[a]pyrene (3-OH-BP), althoughsmall amounts of 9-hydroxybenzo[a]pyrene, quinones and trans-9,10-dihydro-9,10-dihydroxybenzo[a]-pyrene(BP-9,10-diol) were also present. The major organic solvent-extractablemetabolites found in the extracellular culture medium were BP-7,8-dioland BP-9,10-diol, with smaller quantities of unconjugated phenolsand quinones. The major intracellular and extracellular water-solublemetabolites of BP were conjugated with glucuronide (primarily3-OH-BP and several BP-quinones), and to a lesser extent withsulfate (primarily BP-7,8-diol). Both intracellular and extracellularmetabolism of organic solvent-extractable and water-solubleconjugates was significantly inhibited by ellagic acid in adose-dependent manner. The intracellular enzyme-mediated bindingof BP to mouse keratinocyte DNA was also largely inhibited ina dose-dependent fashion by ellagic acid. Our results indicatethat cultured primary mouse keratinocytes offer a useful modelsystem for studying factors affecting the metabolic activationand detoxification of polycyclic aromatic hydrocarbon carcinogensin the epidermis, and that polyphenolic compounds such as ellagicacid may prove useful in modulating the risk of cutaneous cancerthat results from exposure to these environmental chemicals.     

DNA adduct formation of aristolochic acid I and II in vitro and in vivo

Aristolochic acid I (AA I) and aristolochic acid II (AA II),the two main ingredients of the carcinogenic plant extract aristolochicacid (AA), are metabolized to reactive intermediates which bindcovalently to DNA in vitro and in vivo. DNA adduct formationwas analysed by the ³²P-postlabelling assay. In in vitro incubationswith rat liver 9000 g supernatant (S9) and calf thymus DNA (CT-DNA),AA I showed an identical pattern of DNA adducts on thin-layerchromatograms under aerobic and anaerobic conditions, whereasAA II gave rise to DNA adduct formation only anaerobically.The anaerobically obtained DNA adduct pattern by AA II in vitrowas similar to the AA I adduct patterns. Aristolactams I andII, the metabolites of AA I and AA II formed under anaerobicconditions, did not form DNA adducts in the presence of S9 mixand CT-DNA. Incubations with xanthine oxidase, known to enzymaticallyreduce aromatic nitro groups, also activated AA I and AA IIto reactive intermediates, producing almost identical adductpatterns as obtained by S9 mix-mediated metabolism. Activationof AA I by S9 mix in the presence of poly(dG) resulted in theformation of two adducts, one of which was shown to be chromatographicallyindistinguishable from an adduct obtained by reaction with CT-DNA.For the in vivo studies AA I and AA II were administered orallyto male Wistar rats, and DNA from liver, brain, oesophagus,stomach lining, forestomach lining, kidney and bladder was analysedfor DNA adducts by ³²P-postlabelling. The adduct patterns inDNA from forestomach and kidney—target tissues of AA—andDNA from non-target tissues like stomach lining and liver weresimilar to the patterns obtained from the in vitro incubations.In the bladder (also a target tissue) only AA II gave rise toDNA adduct formation. These findings suggest that DNA adductformation by AA I and AA II does not directly correlate withthe initiation of the carcinogenic process and subsequent tumourformation in target tissues in the rat.     

Reaction of DNA with a mutagenic 3-N, N-acetoxyacetylamino-4, 6-dimethyldipyrido[1, 2-a:3', 2'-d]imidazole (N-AcO-AGlu-P-3) related to glutamic acid pyrolysates

3-Amino-4,6-dimethyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-3),an analog amine of the potent genotoxic Glu-P-1 isolated froma glutamic acid pyrolysate, has been chemically synthesized.Glu-P-3 was found much more mutagenic than Glu-P-1 to S. typhimuriumTA 98 and TA 100 with S-9 mix. 3-N,N-acetoxyacetylamino-4,6-dimethyldipyrido[1,2-a:3',2'-d]imidazole (N-AcO-AGIu-P-3), a possible metabolite of Glu-P-3,binds covalently to the C-8 position of guanine residues inDNA. The binding induces large conformational changes of themacromolecule.     

Anti-mutagenesis and anti-promotion by apigenin, robinetin and indole-3-carbinol

We assessed the anti-mutagenic and anti-promotion propertiesof two flavones, apigenin and robinetin, and of indole-3-carbinol,because these compounds have been reported in vegetables, theconsumption of which has been associated with reduced ratesof cancer. However, the active components of these foods andtheir effects on carcinogenesis have not been established. Anti-mutagenicitywas determined in the Salmonella typhimurium assay by measuringthe effects of the test compounds on bacterial mutagenesis inducedby methyl-nitrosourea (MNU), methyl-n-nitro-N-nitrosoguanidine(MNNG), benzo[a]pyrene (BaP) or 2-aminoanthracene (2-AA). Inclusionof apigenin resulted in a 62% and a 43% inhibition of mutagenicitywith 13 nmol of 2-AA and 30 nmol BaP respectively. Robinetincaused an 87% inhibition of mutagenicity by 2-AA, but indole-3-carbinolhad little or no effect on the mutagenicity of any of the compounds.None of the three compounds inhibited mutagenesis by MNU orMNNG and none were mutagenic or toxic when tested in the absenceof mutagenic compounds at doses up to 20 µg/plate. Anti-promotionproperties were assessed by measuring the effects of apigenin,robinetin and indole-3-carbinol on induction of ornithine decarboxylaseactivity (ODC) in mouse epidermis by 17 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA). Pretreatment of the skin half an hourbefore TPA with apigenin, robinetin, butylated hydroxyanisole,13-cis-retinoic acid (all at 50 µol) or di-fluoromethylornithine(1.6 µmol) inhibited ODC induction at 6 h after TPA by67–80%. Pretreatment with 50 µmol indole-3-carbinolcaused a 78% elevation in the TPA induction at this time. Doseresponse measurements were conducted with apigenin, indole-3-carbinoland robinetin. Inhibition by 30–90% of TPA-induced ODCwas observed at 6 h after TPA in mice pre-treated with 12.5–100µmol apigenin. Pretreatment with 37.5 or 50 µmolindole-3-carbinol or 0.5, 12.5 or 25 µmol robinetin resultedin elevated induction of epidermal ODC by TPA at 6 h after TPA.However, treatment with 50 or 100 µmol robinetin diminishedODC induction at 6 h after TPA. Treatment with 100 µmolapigenin or 50 or 100 µmol indole-3-carbinol in non-TPA-treatedmouse skin caused elevations in epidermal ODC. In comparingthe time course of ODC induction, indole-3-carbinol (50 µmol)pretreatment shifted the induction of epidermal ODC to earliertimes, in addition to elevating ODC induction by TPA. However,apigenin (50 µmol) pretreatment inhibited TPA-inducedODC activity at 4, 6 and 8 h, indicating no shift in ODC induction.In conclusion, indole-3-carbinol showed no potential for inhibitionof mutagenesis in the present study and presented potentialfor enhancement of promotion. In contrast, the potential ofapigenin and robinetin as inhibitors of the initiation and promotionphases of carcinogenesis merits further study.     

Organospecific activation of azathioprine in mice: role of liver metabolism in mutation induction

The organ-specific mutagenicity of azathioprine was examinedby means of the intrasangulneous host-mediated assay in mice,combined with the D7 strain of Saccharomyces cerevisiae To assayfor changes in the frequency of mitotic gene conversion, mitoticcrossing-over and point reverse mutation frequency, a singlei.p. dose of azathioprine was administered. Kidney-mediatedmutagenicity was significantly enhanced. The ability of liver,kidney and lung S9 fractions (from Na-phenobarbital + ß-naphthoflavoneinduced mice) to activate azathioprine into genotoxic intermediateswas also evaluated in vitro by incubating organ homogenateswith S.cerevisiae cells as a target organism. Organ-specificactivation was demonstrated only in the liver. The relativerole of liver metabolism in the induction of mutations and tumorinduction was investigated in in vivo experiments with partiallyhepatectomized mice. The data demonstrated that liver affectsboth kidney- and lung-mediated mutagenicity and indicated thathepatic metabolism can contribute mutagenic metabolites forcancer initiation by azathioprine.     

Disposition of the naturally occurring antimutagenic plant phenol, ellagic acid, and its synthetic derivatives, 3-O-decylellagic acid and 3, 3'-di-O-methylellagic acid in mice

The effect of ellagic acid and some of its more lipophilk derivativeson the mutagenicity of (? )-7ß, 8-di-hydroxy-9 10-epoxy-7,8, 910-etrahydrobenz[a]pyrene was examined in Salmonella typhimuriumTA100. Ellagic acid, 3, 3' -di-O-methylellagic add, 4, 4' -di-O-methylellagicacid and 3-O-decyiellagic acid were found to have approximatelyequal antimutagenic activity. The tissue distribution and eliminationof ellagic add, 3, 3' -di-O-methyleCagic add and 3-O-decylellagicacid were examined in CD-I mice. Little or no ellagic acid (<1 nmol/g) was found in blood, lung or liver after the oral administrationby gavage of 300 µunol of ellagic acid per kg body weightor after feeding 1% of ellagic acid in the diet for 1 week.Following the i.p. administration of 120 µmol/kg of ellagicacid, the blood and lung levels of ellagic acid were 15–20nmol/g at 30 min after the dose, and the concentrations of ellagicacid decreased to 1–3 nmol/g at 6–8 h after thedose. A portion of the administered i.p. dose precipitated inthe abdominal cavity. After i.v. administration, ellagic acidwas eliminated very rapidly from blood, lung and liver, and 70% of the administered dose was recovered in the urine andfeces as free ellagic acid and its conjugates. At 2 h afteran i.v. injection of 60 µ/kg of ellagic add, 46% of thedose was recovered in the urine as ellagic acid and its conjugates.Of this amount, about half was excreted as free ellagic acidand half was excreted as conjugates. An additional 25% of thedose was recovered in the feces (mostly as free ellagic acid)after 7 h. The disposition of 3, 3' -di-O-methylelIagic acidor 3–O-decyIellagic acid after i.v. administration (32µmol;sol;kg) was examined and compared to the dispositionof the same i.v. dose of ellagic acid. The concentrations ofellagic acid, 3,3' -di-O-methylellagic acid and 3-O-decytellagicadd decreased rapidly in the blood, liver and lung, but theconcentrations of 3-O-decylellagic add in the lung throughoutthe experimental period (2–360 min) was on average 20-to 40-fold higher than the corresponding average concentrationsof ellagic acid or 3, 3' -di-O-methylellagic acid.     

Induction of mutagenesis and transformation by the extract of Alernaria alternata isolated from grains in Linxian, China

Alternaria alternata is commonly found in the grain in areasof high incidence of oesophageal cancer and is a suspected cancer-causingfactor in Linxian, China. In this study, this fungus was isolatedfrom corn in Linxian and cultured. The extract of this funguswas shown to induce 6-thioguanine-resistant mutants in V79 cellsand cause transformation of NIH/3T3 mouse fibroblast cells.Metabolic activation does not seem to be required for theseactivities. The mutagenic and transforming activities of theextract of A. alternata suggest that this fungus may be a factorin the etiology of oesophageal cancer in Linxian.     

Inhibition of aflatoxin B1 mutagenesis in Salmonella typhimurium and DNA damage in cultured rat and human tracheobronchial tissues by ellagic acid

Ellagic acid (EA), a plant phenol found in various fruits andnuts, was examined for its ability to inhibit aflatoxin B₁ (AFB₁)mutagenesis in strain TA 100 of Salmonella typhimurium. In thepresence of rat liver S-9 microsomal preparation, EA (1.5 µg/plate)inhibited the number of mutations induced by AFB, (0.5 µg/plate)by 50%. EA at a dose of 1000 µg/plate inhibited the mutationfrequency by >90%. EA was also tested for its ability toinhibit the DNA binding and adduct formation of AFB₁ in culturedexplants of rat trachea and human tracheobronchus. Explantswere incubated in medium containing EA at concentrations of10, 50 and 100 µM for 16 h foUowed by the addition of1 µM [₃H]AFB₁ and EA for 24 h. DNA was isolated by phenolextraction and hydroxylapatite chromatography. EA caused a dose-dependentinhibition in the covalent binding of AFB₁ to the DNA of boththe rat trachea (9—57% inhibition) and human tracheobronchus(24—79% inhibition). After acid hydrolysis of the isolatedDNA, the AFB₁—DNA adducts were separated by h.p.l.c. Intissues from both species, the major AFB₁—DNA adductswere AFB₁-N⁷-Gua [8,9-dihydro-8-(N⁷-guanyl)-9-hydroxyAFB₁] andAFB₁-N⁷-FaPyr (major) [8,9-dihydro-8- (2, 6-diamino-4-oxo-3,4-dihydro-pyrimid-5-ylformamido)-9-hydroxyAFB₁], and the formation of these adductswas reduced by 28—76% in the presence of EA. These dataindicate that EA has the potential to act as a naturally occurringinhibitor of AFB₁-related respiratory damage in rats and inhumans.     

32P-Postlabeling analysis of DNA adducts in rat stomach with 1-nitrosoindole-3-acetonitrile, a direct-acting mutagenic indole compound formed by nitrosation

Modification of DNA by a direct-acting mutagen, 1-nitrosoindole-3-acetonitrile,which is formed from indole-3-acetonitrile upon nitrite treatment,was investigated. ³²P-Postlabeling analysis clearly demonstratedthe formation of DNA adducts in the stomach of rats after intragastricadministration of 1-nitrosoindole-3-acetonitrile. The levelof DNA adducts in both the forestomach and glandular stomach2 h after administraction of 100 mg/kg body weight of the compoundwas about one adduct per 10⁷nucleotides. The DNAs of the forestomachand glandular stomach gave six common spots on two-dimensionalchromatography, three of which were also produced by in vitroreaction of this compound with DNA. Thus, 1-nitrosoindole-3-acetonitrilecan form DNA adducts in vivo and in vitro. No DNA adducts weredetected after treatment with the non-nitrosated compound indole-3-acetonitrileboth in vivo and in vitro. These results suggest that 1-nitrosoindole-3-acetonitrilehas the in vivo tumor initiating activity in the stomach.     

Induction of bacterial mutations by aminopyrazoles, compounds which cause mammary cancer in rats

An aminopyrazole PD 71627 (5-amino-l, 3-dimethyl-1H-pyrazol-4-yl)(2-fluorophenyl)methanone, and two amide derivatives, PD 108298,N-[4-(2-flurobenzyD-13-dimethyl-1H/-pyrazol-5-yl]-2-([3K2-methyl-1-piperidmyI)-propyl]amino)acetamide-(Z)-2-butanedioate (1: 2), and PD 109394, 2-(di-ethylamino)-N-(4-(2-fluorobenzoyI)-l,3-dimethyl-1H-5-pyrazol-5-yl]acetamide hydrochloride, proposedneuroleptic drugs, were found to elicit mammary adenocarcinomasin male rats after 13 weeks of treatment. These compounds wereassess-ed for their ability to induce His⁺ revertants (rev)in five strains of Salmonella typhimurium (TA98, TA100, TA1535,TA1537 and TA1538) in the presence and absence of S9 activation.All were found to be potent mutagens in TA98 and TA100 aftera 20 min pre-incubation with Aroclor 1254-induced rat liverS9. However, the activity of the aminopyrazole PD 71627 wasmuch greater than the amide derivatives, PD 108298 or PD 109394,with activity of 11 800 rev/µmol, 670 rev/µmol,and 230 rev/µmol respectively in TA100, the strain showingthe greatest response. A comparison of liver S9 fractions fromrats untreated or pretreated with phenobarbital (PB) or Aroclor1254 showed that S9 from animals pretreated with PB providedthe greatest activation capability for the aminopyrazole PD71627 (59 300 rev/µmol in TA100). Three structural analogsof the aminopyrazole PD 71627, two without the amine and onewith a methyl substi-tuent on the amine, were compared withPD 71627 for in-duction of revertants in TA100 and TA98. Thecompounds without the amine had no mutagenk activity while themethyl derivative induced 3100 rev/µmol in TA100 afterpreincuba-tion with Aroclor 1254-induced S9. This confirmedthat the amine on the pyrazole ring was required for mutagenkac-tivity. The results of these studies support the hypothesisthat these compounds cause cancer in animals as a result ofDNA damage.     

N-methyl-N''-nitro-N-nitrosoguanidine induced genetic change during the meiotic cell cycle in Saccharomyces cerevisiae: an absence of S-phase specificity

In the yeast Saccharomyces cerevisiae enhanced nuclear mutagenesiswas found during mitotic DNA synthesis after treatment withN-methyl-N'-nitro-N-nitrosoguanidine (MNNG), but not duringmeiotic DNA synthesis. Further experiments found no significantvariation in the mol. wt. of DNA from control or MNNG-treatedsamples during meiosis after alkaline sucrose gradient analysis.Using tritiated MNNG no variation in uptake of MNNG by meioticcells was found, but enhanced binding of label from [³H]MNNGto mitochondrial DNA was detected during meiotic DNA synthesiswhen mitochondrial DNA synthesis occurred. In contrast, no enhancedbinding of label from [³H]MNNG to the nuclear DNA during meioticDNA synthesis was found.     

A demonstration of the in vitro bacterial mutagenicity of procarbazine, using the microtitre fluctuation test and large concentrations of S9 fraction

The anti-neoplastic agent procarbazine is genetically activein a variety of short term mutagenicity tests, and it also possessescarcinogenic and teratogenic potential. This compound has consistentlyyielded false negative results in in vitro microbial mutagenicitytests in the presence and absence of mammalian metabolic activation.In this study, procarbazine was not mutagenic in standard andpreincubation Ames tests using large S9 concentrations and bacterialtest strains devoid of the rfa mutation. The microtitre fluctuationtest is a sensitive technique for the detection of bacterialmutagens. Using this assay, procarbazine proved to be a bacterialmutagen after in vitro metabolic activation at concentrationsas low as 200 µg/ml. Activity was dependent upon liverS9-concentrations which were higher than those usually attainedwithin agar assays, and was only observed when such fractionswere derived from aroclor-treated or phenobarbitone plus ß-naphthoflavone treated rat livers. The excision-repair proficientstrain E. coli WP2 was equally, if not more, sensitive thanthe repair deficient strain E. coli WP2 uvrA. Furthermore, thedifferential mutagenic effects obtained using S. typhimuriumstrains TA1535, TA1530 and his G46 indicated that the absenceof an rfa mutation was essential for procarbazine mutagenesis.Procarbazine was also mutagenic for E. coli D494 in a forwardmutation fluctuation assay measuring resistance to ampicillin.In this assay lower concentrations of S9-fraction were sufficient,reflecting the increased sensitivity of the test strain towardsthe mutagemc metabolites of the drug. In conclusion, the resultssuggest that rat liver S9-fraction contains only low levelsof enzymes capable of activating procarbazine to a mutagen.This may be a result of rapid breakdown during storage, or dueto intrinsically low levels in rat liver extracts. Inducingagents appear to increase these levels. The low concentrationsof mutagenic species formed in vitro, may only be detectableusing a highly sensitive assay such as the microtitre fluctuationtest.     

Effect of sulfite on the covalent reaction of benzo(a)pyrene metabolites with DNA

Sulfur dioxide (SO₂) potentiates the carcinogenicity of polycyclicaromatic hydrocarbons. To investigate the mechanism of SO₂ cocarcinogenesis,the effect of sulfite, the hydrated form of SO₂, on the covalentreaction of benzo(a)pyrene (BaP) metabolites with DNA in vitrowas measured. (¹⁴C)BaP was incubated with rat lung or liverpost-mitochondrial supernatant (S9), an NADPH generating system,calf thymus DNA and sodium sulfite (0–20 mM). In the presenceof lung S9, covalent reaction increased linearly from 0.66 to1.20 pmol BaP metabolites per mg DNA with increasing sulfiteconcentrations. Addition of sulfite to rat liver S9 also increasedBaP-DNA adduct formation with BaP-DNA adducts increasing from80 to 120 pmol per mg DNA. Sulfite altered the amount and patternof BaP metabolites formed by either lung or liver enzyme preparations.BaP was metabolized more extensively and the amount of watersoluble BaP metabolites formed increased significantly withsulfite present. With lung S9, the amount of BaP-tetrols, diols,and phenols increased slightly. With liver S9, diol and phenolformation was significantly lower while tetrol formation wasunchanged. Incubation of rat lung S9 with sulfite resulted information of glutathione S-sulfonate (GSSO₃H), a known inhibitorof glutathione S-transferases mediating the conjugation of glutathione(GSH) and BaP epoxides. Our results suggest that sulfite may,by altering the overall metabolic activation and detoxicationof BaP, or by reacting directly with DNA, subsequently affectthe covalent reaction of BaP metabolites with DNA. These areoffered as possible mechanisms to explain the cocarcinogeniceffect of SO₂.     

Procarcinogen activation by rat and human mammary extracts

Sprague-Dawley rat mammary gland is extremely sensitive to tumorigenesisby single or multiple doses of several poly-cydk aromatic hydrocarbons.We obtained quantitative data on the in vitro mutagenic activationof several procarcinogens by 9000 g supernatant fraction (S9)from rat mammary gland using the Ames test. Mutagenic activationwas shown to be dependent on a nicotinamide adenine dinucleotidephosphate (NADPH) generating system. An S9 preparation frommammary tissue of lactating Sprague-Dawley rats was shown toactivate 2-aminoanthracene (2-AA). A polychlorinated biphenylmixture of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) givento rats greatly raised the specific activity (rever-tant TA98colonies/mg S9 protein) of the mammary tissue using 2-AA asa test carcinogen, and permitted detection of 2, 4-diaminoanisole(DAA) and 2, 7-diaminofluorene (DAF) activation. Procarcinogens2-aminofluorine (2-AF), benzo[a]-pyrene (BP) and aflatoxin (AFL)Bl were not detectably activated by mammary gland. Mutagenesisproduced in mammary S9 activation of 2-AA, DAA or DAF was significantlyinhibited by alpha-naphthoflavone (NF) but was inhibited minimallyby metyrapone (MP). Human mammary tumor cell lines (734B, SkBr3,MDA-MD-330) possessed inducible procarcinogen metabolizing activitiessimilar to those found in S9 of rat mammary tissue. We demonstrateda simple and convenient use of the Ames test to characterizeactivation of many potential mutagens and carcinogens for mammarygland. When a test compound such as 2-AA was used, selectiveenzyme induction and inhibition was demonstrated.     

靶向HRP/IAA自杀基因系统联合IL-12基因治疗Lewis肺癌的研究

目的:观察人端粒酶逆转录酶(hTERT)启动子驱动辣根过氧化物酶(HRP)/吲哚乙酸(IAA)系统联合白细胞介素-12(IL-12)基因对小鼠Lewis肺癌的疗效。方法:建立Lewis肺癌移植瘤模型,随机分为对照组、HRP组、IL-12组和联合组,分别给予AdCMVGFP、AdhTERTHRP、AdCMVmIL-12单一或联合注射,然后腹腔注射IAA,观察肿瘤生长情况。蛋白质印迹法和ELISA检测瘤组织HRP和IL-12表达;免疫组化检测瘤组织内CD4+、CD8+淋巴细胞浸润情况。结果:与对照组、单一治疗组相比,联合组小鼠肿瘤生长受到显著抑制(联合组vs对照组:P=0.000,9 d;联合组vsHRP组:P=0.005,15 d;联合组vsIL-12组:P=0.046,12 d),生存期显著延长(联合组vs对照组:χ2=9.529,P=0.002;联合组vsHRP组:χ2=9.039,P=0.003;联合组vsIL-12组:χ2=8.595,P=0.003),肿瘤组织广泛坏死,CD4+、CD8+淋巴细胞浸润显著增多。结论:IL-12基因治疗可诱导宿主产生抗肿瘤免疫应答,与靶向HRP/IAA自杀基因系统联合具有...